Modified Gene Silencing

ABSTRACT

This invention relates to methods of controlling gene expression or gene suppression in eukaryotic cells. One aspect of this invention includes modifying the degree of silencing of a target gene by use of a modified suppression element. Another aspect includes providing a eukaryotic cell having a desired phenotype resulting from transcription in the eukaryotic cell of a modified suppression element. Also provided are transgenic eukaryotic cells, transgenic plant cells, plants, and seeds containing modified suppression elements, and useful derivatives of such transgenic plant cells, plants, or seeds, such as food or feed products.

PRIORITY CLAIMS AND REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/749,553, filed on Mar. 30, 2010, which is a divisional of and claimsbenefit of priority to U.S. patent application Ser. No. 11/674,207,filed on Feb. 13, 2007, which claims priority to U.S. Provisional PatentApplication No. 60/772,614, filed on Feb. 13, 2006, all of which areincorporated by reference in their entirety herein.

FIELD OF THE INVENTION

Disclosed herein are methods of controlling gene expression ineukaryotic cells.

BACKGROUND OF THE INVENTION

Methods to control gene expression by silencing or suppressing a targetgene include use of antisense, co-suppression, and RNA interference.Anti-sense gene suppression in plants is described by Shewmaker et al.in U.S. Pat. Nos. 5,107,065, 5453,566, and 5,759,829. Gene suppressionin bacteria using DNA which is complementary to mRNA encoding the geneto be suppressed is disclosed by Inouye et al. in U.S. Pat. Nos.5,190,931, 5,208,149, and 5,272,065. RNA interference or double-strandedRNA-mediated gene suppression has been described by, e. g., Redenbaughet al. in “Safety Assessment of Genetically Engineered Fruits andVegetables”, CRC Press, 1992; Chuang et al. (2000) PNAS, 97:4985-4990;and Wesley et al. (2001) Plant J., 27:581-590.

In some cases, total or maximal silencing or suppression is desired, forexample, where a suppression element is designed to suppress a pathogentarget gene in order to achieve maximal protection of the host of thatpathogen. However, complete suppression of a gene is not alwayspreferred, for example, where complete suppression decreases viabilityor robustness of the organism in which the gene is silenced. In somecases, a desired phenotype (e. g., a particular level of a metabolite ora particular combination of traits) is associated with a specific levelof suppression of a target gene. Thus, it is useful to be able to modifythe level of silencing of a target gene or genes by a suppressionelement.

SUMMARY OF THE INVENTION

This invention discloses methods of controlling gene expression or genesuppression in eukaryotic cells. One aspect of the invention provides amethod of modifying the degree of silencing of a target gene, includingtranscribing in a eukaryotic cell a modified suppression element,thereby obtaining a modified degree of silencing of the target gene,relative to a reference degree of silencing obtained through thetranscription in the eukaryotic cell of a reference suppression elementthat corresponds to the target gene.

A second aspect of the invention provides a method of providing aeukaryotic cell having a desired phenotype resulting from transcriptionin the eukaryotic cell of a modified suppression element, including (a)providing a range of modified suppression elements, wherein eachmodified suppression element includes a fragment of a referencesuppression element; (b) separately introducing each of the range ofmodified suppression elements into a eukaryotic cell, thereby producinga plurality of transgenic eukaryotic cells; (c) transcribing in each ofthe transgenic eukaryotic cell the modified suppression element thereinintroduced, and observing the resulting phenotype resulting from thetranscribing; and (d) selecting from the plurality of transgeniceukaryotic cells at least one eukaryotic cell having the desiredphenotype.

The invention further provides transgenic eukaryotic cells, andorganisms containing such cells, having a phenotype resulting fromsuppression of a target gene by transcription in the eukaryotic cell ofa modified suppression element. Further provided are transgenic plantcells, plants, and seeds containing modified suppression elements of theinvention, and useful derivatives of such transgenic plant cells,plants, or seeds, such as food or feed products. Other specificembodiments of the invention are disclosed in the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts a method for mapping silencing efficacy ofa suppression element, useful in designing or selecting a modifiedsuppression element, as described in Example 1.

FIG. 2 depicts results of the experiments described in Example 2 as aplot of 18:1 fatty acid levels as a function of size of the suppressionelement.

FIG. 3 depicts results of the experiments described in Example 2, as aplot of 18:1 fatty acid levels as a function of size of the suppressionelement.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the manufacture or laboratory procedures described beloware well known and commonly employed in the art. Conventional methodsare used for these procedures, such as those provided in the art andvarious general references. Unless otherwise stated, nucleic acidsequences in the text of this specification are given, when read fromleft to right, in the 5′ to 3′ direction. Where a term is provided inthe singular, the inventors also contemplate aspects of the inventiondescribed by the plural of that term. The nomenclature used herein andthe laboratory procedures described below are those well known andcommonly employed in the art. All of the United States Patents andUnited States Patent Application Publications cited in the descriptionof the invention are herein incorporated by reference in their entirety.Where there are discrepancies in terms and definitions used in citationsthat are incorporated by reference, the terms used in this applicationshall have the definitions given herein. Other technical terms usedherein have their ordinary meaning in the art that they are used, asexemplified by a variety of technical dictionaries. The inventors do notintend to be limited to a mechanism or mode of action. Reference theretois provided for illustrative purposes only.

Method of Modifying Gene Silencing

In one aspect, this invention provides a method of modifying the degreeof silencing of a target gene, including transcribing in a eukaryoticcell a modified suppression element, thereby obtaining a modified degreeof silencing of the target gene, relative to a reference degree ofsilencing obtained through the transcription in the eukaryotic cell of areference suppression element that corresponds to the target gene.

Eukaryotic Cells

The eukaryotic cell in which the suppression elements are to betranscribed can include any eukaryotic cell or cells. In preferredembodiments, the eukaryotic cell is selected from an animal cell and aplant cell. The eukaryotic cell can be a discrete cell (e. g., a plant,yeast, fungal, insect, or mammalian cell grown under cell cultureconditions), a cell in undifferentiated tissue (e. g., a callus ofundifferentiated plant cells), a cell in differentiated tissue, or acell in an intact multicellular organism (such as a plant or an animalof any age or growth or reproductive stage).

Target Genes

The target gene to be silenced can include any gene, and can includemultiple genes. The target gene can include a gene endogenous to theeukaryotic cell in which the suppression element is transcribed, such asa gene or genes native to the eukaryotic cell, or a transgene in atransgenic eukaryotic cell. The target gene can include a gene exogenousto the eukaryotic cell in which the suppression element is transcribed,for example, a gene native to a pest or pathogen or to a symbiont (e.g., a endobiont or an endocytobiont) of the eukaryotic cell.

The target gene can include a single gene or part of a single gene thatis targetted for suppression, or can include, e. g., multipleconsecutive segments of a target gene, multiple non-consecutive segmentsof a target gene, multiple alleles of a target gene, or multiple targetgenes from one or more species. In some embodiments, the target geneincludes one contiguous nucleotide sequence. In other embodiments, thetarget gene includes non-contiguous nucleotide sequences, e. g.,non-contiguous segments of a single mRNA transcript or non-contiguoussegments of a native DNA nucleotide sequence (which can include codingDNA or non-coding DNA or a combination of both).

The target gene can be include nucleotides in translatable (coding)sequence, or nucleotides in non-coding sequence (such as non-codingregulatory sequence), or nucleotides in both coding and non-codingsequence. The target gene can include at least one eukaryotic targetgene, at least one non-eukaryotic target gene, or both. A target genecan include any sequence from any species (including, but not limitedto, non-eukaryotes such as bacteria and viruses; fungi; plants,including monocots and dicots, such as crop plants, ornamental plants,and non-domesticated or wild plants; prokaryotic or eukaryotic symbiontsincluding intercellular symbionts (endobionts), intracellular symbionts(endocytobionts), and external symbionts (ectosymbionts); andinvertebrates (e. g., arthropods, annelids, nematodes, and molluscs);and vertebrates (e.g., amphibians, fish, birds, domestic or wildmammals, and even humans).

Non-limiting examples of a target gene include non-translatable(non-coding) DNA, such as, but not limited to, 5′ untranslated regions,promoters, enhancers, or other non-coding transcriptional regions, 3′untranslated regions, terminators, and introns. Target genes can alsoinclude genes encoding microRNAs, small interfering RNAs, RNA componentsof ribosomes or ribozymes, small nucleolar RNAs, and other non-codingRNAs (see, for example, non-coding RNA sequences described byGriffiths-Jones et al. (2005) Nucleic Acids Res., 33:121-124, andnon-coding RNAs which lack long open reading frames and function asriboregulators, as described by Erdmann et al. (2001) Nucleic AcidsRes., 29:189-193). One specific example of a target gene includes amicroRNA recognition site (i.e., the site on an RNA strand to which amature miRNA binds and induces cleavage). Another specific example of atarget gene includes a microRNA precursor sequence, that is, the primarytranscript encoding a microRNA, or the RNA intermediates processed fromthis primary transcript (e. g., a nuclear-limited pri-miRNA or apre-miRNA which can be exported from the nucleus into the cytoplasm).See, for example, Lee et al. (2002) EMBO Journal, 21:4663-4670; Reinhartet al. (2002) Genes & Dev., 16:161611626; Lund et al. (2004) Science,303 :95-98; and Millar and Waterhouse (2005) Funct. Integr Genomics,5:129-135. Target microRNA precursor DNA can be native to the transgenicplant of the invention, or can be native to a pest or pathogen of thetransgenic plant. Target DNA can also include translatable (coding)sequence for genes encoding transcription factors and genes encodingenzymes involved in the biosynthesis or catabolism of molecules (suchas, but not limited to, amino acids, fatty acids and other lipids,sugars and other carbohydrates, biological polymers, and secondarymetabolites including alkaloids, terpenoids, polyketides, non-ribosomalpeptides, and secondary metabolites of mixed biosynthetic origin). Atarget gene can be a native gene targetted for suppression, with orwithout concurrent expression (or suppression) of an exogenoustransgene, e.g.,, by including a gene expression (or suppression)element in the same or in a separate recombinant DNA construct. Forexample,a native gene can be replaced with an exogenous transgenehomologue.

In one embodiment, the suppression element is transcribed in atransgenic plant, and suppresses a target gene which is exogenous to thehost plant but endogenous to a plant pest or pathogen (e. g., viruses,bacteria, fungi, and invertebrates such as insects, nematodes, andmolluscs). Thus, in one embodiment the target gene is selected toprovide resistance to a plant pest or pathogen, for example, resistanceto a nematode such as soybean cyst nematode or root knot nematode or toa pest insect such as corn rootworm. Thus, target genes also includeendogenous genes of plant pests and pathogens. Pest invertebratesinclude, but are not limited to, pest nematodes (e. g., cyst nematodesHeterodera spp. especially soybean cyst nematode Heterodera glycines,root knot nematodes Meloidogyne spp., lance nematodes Hoplolaimus spp.,stunt nematodes Tylenchorhynchus spp., spiral nematodes Helicotylenchusspp., lesion nematodes Pratylenchus spp., ring nematodes Criconema spp.,and foliar nematodes Aphelenchus spp. or Aphelenchoides spp.), pestmolluscs (slugs and snails), and pest insects (e. g., corn rootworms,Lygus spp., aphids, corn borers, cutworms, armyworms, leafhoppers,Japanese beetles, grasshoppers, and other pest coleopterans, dipterans,and lepidopterans). Plant pathogens include fungi (e. g., the fungi thatcause powdery mildew, rust, leaf spot and blight, damping-off, root rot,crown rot, cotton boll rot, stem canker, twig canker, vascular wilt,smut, or mold, including, but not limited to, Fusarium spp., Phakosporaspp., Rhizoctonia spp., Aspergillus spp., Gibberella spp., Pyriculariaspp., Alternaria spp., and Phytophthora spp.), bacteria (e. g., thebacteria that cause leaf spotting, fireblight, crown gall, and bacterialwilt), mollicutes (e. g., the mycoplasmas that cause yellows disease andspiroplasmas such as Spiroplasma kunkelii, which causes corn stunt), andviruses (e. g., the viruses that cause mosaics, vein banding, flecking,spotting, or abnormal growth). See also G. N. Agrios, “Plant Pathology”(Fourth Edition), Academic Press, San Diego, 1997, 635 pp., fordescriptions of fungi, bacteria, mollicutes (including mycoplasmas andspiroplasmas), viruses, nematodes, parasitic higher plants, andflagellate protozoans, all of which are plant pests or pathogens. Seealso the continually updated compilation of plant pests and pathogensand the diseases caused by such on the American PhytopathologicalSociety's “Common Names of Plant Diseases”, compiled by the Committee onStandardization of Common Names for Plant Diseases of The AmericanPhytopathological Society, 1978-2005.

Non-limiting examples of fungal plant pathogens of particular interestinclude Phakospora pachirhizi (Asian soy rust), Puccinia sorghi (corncommon rust), Puccinia polysora (corn Southern rust), Fusarium oxysporumand other Fusarium spp., Alternaria spp., Penicillium spp., Pythiumaphanidermatum and other Pythium spp., Rhizoctonia solani, Exserohilumturcicum (Northern corn leaf blight), Bipolaris maydis (Southern cornleaf blight), Ustilago maydis (corn smut), Fusarium graminearum(Gibberella zeae), Fusarium verticilliodes (Gibberella moniliformis), F.proliferatum (G. fujikuroi var. intermedia), F. subglutinans (G.subglutinans), Diplodia maydis, Sporisorium holci-sorghi, Colletotrichumgraminicola, Setosphaeria turcica, Aureobasidium zeae, Phytophthorainfestans, Phytophthora sojae, Sclerotinia sclerotiorum, and thenumerous fungal species provided in Tables 4 and 5 of U.S. Pat. No.6,194,636, incorporated by reference.

Non-limiting examples of bacterial pathogens include Pseudomonas avenae,Pseudomonas andropogonis, Erwinia stewartii, Pseudomonas syringae pv.syringae, and the numerous bacterial species listed in Table 3 of U.S.Pat. No. 6,194,636, incorporated by reference.

Non-limiting examples of viral plant pathogens of particular interestinclude maize dwarf mosaic virus (MDMV), sugarcane mosaic virus (SCMV,formerly MDMV strain B), wheat streak mosaic virus (WSMV), maizechlorotic dwarf virus (MCDV), barley yellow dwarf virus (BYDV), bananabunchy top virus (BBTV), and the numerous viruses listed in Table 2 ofU.S. Pat. No. 6,194,636, incorporated by reference.

Non-limiting examples of invertebrate pests include pests capable ofinfesting the root systems of crop plants, e. g., northern corn rootworm(Diabrotica barberi), southern corn rootworm (Diabroticaundecimpunctata), Western corn rootworm (Diabrotica virgifera), cornroot aphid (Anuraphis maidiradicis), black cutworm (Agrotis ipsilon),glassy cutworm (Crymodes devastator), dingy cutworm (Feltia ducens),claybacked cutworm (Agrotis gladiaria), wireworm (Melanotus spp., Aeolusmellillus), wheat wireworm (Aeolus mancus), sand wireworm (Horistonotusuhlerii), maize billbug (Sphenophorus maidis), timothy billbug(Sphenophorus zeae), bluegrass billbug (Sphenophorus parvulus), southerncorn billbug (Sphenophorus callosus), white grubs (Phyllophaga spp.),seedcorn maggot (Delia platura), grape colaspis (Colaspis brunnea),seedcorn beetle (Stenolophus lecontei), and slender seedcorn beetle(Clivinia impressifrons), as well as the parasitic nematodes listed inTable 6 of U.S. Pat. No. 6,194,636, incorporated by reference.

Target genes from pests can include invertebrate genes for major spermprotein, alpha tubulin, beta tubulin, vacuolar ATPase,glyceraldehyde-3-phosphate dehydrogenase, RNA polymerase II, chitinsynthase, cytochromes, miRNAs, miRNA precursor molecules, miRNApromoters, as well as other genes such as those disclosed in Table II ofUnited States Patent Application Publication 2004/0098761 A1,incorporated by reference. Target genes from pathogens can include genesfor viral translation initiation factors, viral replicases, miRNAs,miRNA precursor molecules, fungal tubulin, fungal vacuolar ATPase,fungal chitin synthase, enzymes involved in fungal cell wallbiosynthesis, cutinases, melanin biosynthetic enzymes,polygalacturonases, pectinases, pectin lyases, cellulases, proteases,and other genes involved in invasion and replication of the pathogen inthe infected plant. Thus, a target gene need not be endogenous to theplant in which the suppression element is transcribed. A suppressionelement can be transcribed in a plant and used to suppress a gene of apathogen or pest that may infest the plant.

Specific, non-limiting examples of suitable target genes also includeamino acid catabolic genes (such as, but not limited to, the maizeLKR/SDH gene encoding lysine-ketoglutarate reductase (LKR) andsaccharopine dehydrogenase (SDH), and its homologues), maize zein genes,genes involved in fatty acid synthesis (e. g., plant microsomal fattyacid desaturases and plant acyl-ACP thioesterases, such as, but notlimited to, those disclosed in U.S. Pat. Nos. 6,426,448, 6,372,965, and6,872,872), genes involved in multi-step biosynthesis pathways, where itmay be of interest to regulate the level of one or more intermediates,such as genes encoding enzymes for polyhydroxyalkanoate biosynthesis(see, for example, U.S. Pat. No. 5,750,848); and genes encodingcell-cycle control proteins, such as proteins with cyclin-dependentkinase (CDK) inhibitor-like activity (see, for example, genes disclosedin International Patent Application Publication Number WO 05007829A2).Target genes include genes encoding undesirable proteins (e. g.,allergens or toxins) or the enzymes for the biosynthesis of undesirablecompounds (e. g., undesirable flavor or odor components). Thus, oneembodiment of the invention is a transgenic plant or tissue of such aplant that is improved by the suppression of allergenic proteins ortoxins, e. g., a peanut, soybean, or wheat kernel with decreasedallergenicity. Target genes include genes involved in fruit ripening,such as polygalacturonase. Target genes include genes where expressionis preferably limited to a particular cell or tissue or developmentalstage, or where expression is preferably transient, that is to say,where constitutive or general suppression, or suppression that spreadsthrough many tissues, is not necessarily desired. Thus, other examplesof suitable target genes include genes encoding proteins that, whenexpressed in transgenic plants, make the transgenic plants resistant topests or pathogens (see, for example, genes for cholesterol oxidase asdisclosed in U.S. Pat. No. 5,763,245); genes where expression is pest-or pathogen-induced; and genes which can induce or restore fertility(see, for example, the barstar/barnase genes described in U.S. Pat. No.6,759,575).

Suppression Elements

The suppression elements of use in the method can be any suppressionelement (or combination of elements) that, when transcribed in theeukaryotic cell, results in silencing of the target gene. Thesuppression element can be transcribable DNA of any suitable length, andwill generally include at least about 19 to about 27 nucleotides (forexample 19, 20, 21, 22, 23, or 24 nucleotides) for every target gene. Inmany embodiments the suppression element includes more than 23nucleotides (for example, more than about 30, about 50, about 100, about200, about 300, about 500, about 1000, about 1500, about 2000, about3000, about 4000, or about 5000 nucleotides) for every target geneintended to be suppressed. In some preferred embodiments, thesuppression element includes “sense” sequence (i. e., nucleotidesequence that is identical or substantially identical to at least onecontiguous segment of the sequence of the gene targetted forsuppression), or “anti-sense” sequence (i. e., nucleotide sequence thatis complementary or substantially complementary to or that formsWatson-Crick base pairs with at least one contiguous segment of thesequence of the gene targetted for suppression), or both sense andanti-sense sequence. In many preferred embodiments, the modified andreference suppression elements transcribe to at least partiallydouble-stranded RNA. Suitable gene suppression elements are described indetail in U.S. Patent Application Publication 2006/0200878, incorporatedby reference, and can be of any one or more types, including:

(a) DNA that includes at least one anti-sense DNA segment that isanti-sense to at least one segment of the target gene;

(b) DNA that includes multiple copies of at least one anti-sense DNAsegment that is anti-sense to at least one segment of the target gene;

(c) DNA that includes at least one sense DNA segment that is at leastone segment of the target gene;

(d) DNA that includes multiple copies of at least one sense DNA segmentthat is at least one segment of the target gene;

(e) DNA that transcribes to RNA for suppressing the target gene byforming double-stranded RNA and includes at least one anti-sense DNAsegment that is anti-sense to at least one segment of the target geneand at least one sense DNA segment that is at least one segment of thetarget gene;

(f) DNA that transcribes to RNA for suppressing the target gene byforming a single double-stranded RNA and includes multiple serialanti-sense DNA segments that are anti-sense to at least one segment ofthe target gene and multiple serial sense DNA segments that are at leastone segment of the target gene;

(g) DNA that transcribes to RNA for suppressing the target gene byforming multiple double strands of RNA and includes multiple anti-senseDNA segments that are anti-sense to at least one segment of the targetgene and multiple sense DNA segments that are at least one segment ofthe target gene, and wherein the multiple anti-sense DNA segments andthe multiple sense DNA segments are arranged in a series of invertedrepeats;

(h) DNA that includes nucleotides derived from a plant miRNA;

(i) DNA that includes nucleotides of a siRNA;

(j) DNA that transcribes to an RNA aptamer capable of binding to aligand; and

(k) DNA that transcribes to an RNA aptamer capable of binding to aligand, and

DNA that transcribes to regulatory RNA capable of regulating expressionof the target gene, wherein the regulation is dependent on theconformation of the regulatory RNA, and the conformation of theregulatory RNA is allosterically affected by the binding state of theRNA aptamer.

Any of these suppression elements can be designed to suppress more thanone target gene, including, for example, more than one allele of atarget gene, multiple target genes (or multiple segments of at least onetarget gene) from a single species, or target genes from differentspecies.

Where the suppression element includes multiple copies of anti-sense ormultiple copies of sense DNA sequence, these multiple copies can bearranged serially in tandem repeats. In some embodiments, these multiplecopies can be arranged serially end-to-end, that is, in directlyconnected tandem repeats. In some embodiments, these multiple copies canbe arranged serially in interrupted tandem repeats, where one or morespacer DNA segments can be located adjacent to one or more of themultiple copies. Tandem repeats, whether directly connected orinterrupted or a combination of both, can include multiple copies of asingle anti-sense or multiple copies of a single sense DNA sequence in aserial arrangement or can include multiple copies of more than oneanti-sense DNA sequence or of more than one sense DNA sequence in aserial arrangement. Where the suppression element includes multiplecopies, the degree of complementarity can be, but need not be, identicalfor all of the multiple copies.

Generally, the reference suppression element and the modifiedsuppression element are of similar types, e. g., both the referencesuppression element and the modified suppression element include DNAthat transcribes to RNA for suppressing the target gene by formingdouble-stranded RNA and includes at least one anti-sense DNA segmentthat is anti-sense to at least one segment of the target gene and atleast one sense DNA segment that is at least one segment of the targetgene. However, in some embodiments the modified suppression element andreference suppression element may be of different types, e. g., thereference suppression element includes an antisense DNA segment that isanti-sense to one or more segments of the target gene, whereas themodified suppression element includes multiple copies of the same ormodified anti-sense DNA segment.

In those embodiments wherein the suppression element includes either atleast one anti-sense DNA segment that is anti-sense to at least onesegment of the at least one target gene or at least one sense DNAsegment that is at least one segment of the at least one target gene,RNA transcribed from either the at least one anti-sense or at least onesense DNA can become double-stranded by the action of an RNA-dependentRNA polymerase (see, for example, U.S. Pat. No. 5,283,184). Thedouble-stranded RNA can be in the form of a single dsRNA “stem” (regionof base-pairing between sense and anti-sense strands), or can havemultiple dsRNA “stems”. Such multiple dsRNA “stems” can further bearranged in series or clusters to form tandem inverted repeats, orstructures resembling “hammerhead” or “cloverleaf” shapes. Any of thesesuppression elements can further include spacer DNA segments foundwithin a dsRNA “stem” (for example, as a spacer between multipleanti-sense or sense DNA segments or as a spacer between a base-pairinganti-sense DNA segment and a sense DNA segment) or outside of adouble-stranded RNA “stem” (for example, as a loop region separating apair of inverted repeats). In cases where base-pairing anti-sense andsense DNA segment are of unequal length, the longer segment can act as aspacer.

The suppression element can include spacer DNA, which can be virtuallyany DNA (such as, but not limited to, translatable DNA sequence encodinga gene, translatable DNA sequence encoding a marker or reporter gene;transcribable DNA derived from an intron, which upon transcription canbe excised from the resulting transcribed RNA; transcribable DNAsequence encoding RNA that forms a structure such as a loop or stem oran aptamer capable of binding to a specific ligand; spliceable DNA suchas introns and self-splicing ribozymes; transcribable DNA encoding asequence for detection by nucleic acid hybridization, amplification, orsequencing; and a combination of these). Spacer DNA can be found, forexample, between parts of a suppression element, or between differentsuppression elements. In some embodiments, spacer DNA is itself sense oranti-sense sequence of the target gene. In some preferred embodiments,the RNA transcribed from the spacer DNA (e. g., a large loop ofantisense sequence of the target gene or an aptamer) assumes a secondarystructure or three-dimensional configuration that confers on thetranscript a desired characteristic, such as increased stability,increased half-life in vivo, or cell or tissue specificity.

In a further embodiment, the suppression element can include DNA thatincludes nucleotides derived from a miRNA (microRNA), that is, a DNAsequence that corresponds to a miRNA native to a virus or a eukaryote(including plants and animals, especially invertebrates), or a DNAsequence derived from such a native miRNA but modified to includenucleotide sequences that do not correspond to the native miRNA. Aparticularly preferred embodiment includes a suppression elementcontaining DNA that includes nucleotides derived from a viral or plantmiRNA. A further embodiment includes a suppression element with DNAsequence that corresponds to a miRNA that is native to a fungus.

In a non-limiting example, the nucleotides derived from a miRNA caninclude DNA that includes nucleotides corresponding to the loop regionof a native miRNA and nucleotides that are selected from a target genesequence. In another non-limiting example, the nucleotides derived froma miRNA can include DNA derived from a miRNA precursor sequence, such asa native pri-miRNA or pre-miRNA sequence, or nucleotides correspondingto the regions of a native miRNA and nucleotides that are selected froma target gene sequence number such that the overall structure (e. g.,the placement of mismatches in the stem structure of the pre-miRNA) ispreserved to permit the pre-miRNA to be processed into a mature miRNA.In yet another embodiment, the suppression element can include DNA thatincludes nucleotides derived from a miRNA and capable of inducing orguiding in-phase cleavage of an endogenous transcript into trans-actingsiRNAs, as described by Allen et al. (2005) Cell, 121:207-221. Thus, theDNA that includes nucleotides derived from a miRNA can include sequencenaturally occurring in a miRNA or a miRNA precursor molecule, syntheticsequence, or both.

In preferred embodiments, the suppression element is included in arecombinant DNA construct that is useful in producing a transgeniceukaryotic cell. Generally, such a construct includes a promoter that isable to initiate transcription in the eukaryotic cell, and that isoperably linked, directly or with intervening sequences, to thesuppression element. The construct can optionally include a terminatorelement.

Where the recombinant DNA construct is to be transcribed in an animalcell, the promoter element is functional in an animal. Where therecombinant DNA construct is to be transcribed in a plant cell, thepromoter element is functional in a plant. In various embodiments, thepromoter element can include a promoter selected from the groupconsisting of a constitutive promoter, a spatially specific promoter, atemporally specific promoter, a developmentally specific promoter, andan inducible promoter. Where transcription of the construct is to occurin a plant cell, spatially specific promoters can include organelle-,cell-, tissue-, or organ-specific promoters functional in a plant (e.g., a plastid-specific, a root-specific, a pollen-specific, or aseed-specific promoter for expression in plastids, roots, pollen, orseeds, respectively). In many cases a seed-specific, embryo-specific,aleurone-specific, or endosperm-specific promoter is especially useful.Where transcription of the construct is to occur in an animal cell,spatially specific promoters include promoters that have enhancedactivity in a particular animal cell or tissue (e. g., enhanced orspecific promoter activity in nervous tissue, liver, muscle, eye, blood,marrow, breast, prostate, gonads, or other tissues). Temporally specificpromoters can include promoters that tend to promote expression duringcertain developmental stages in an animal or plant's growth orreproductive cycle, or during different times of day or night, or atdifferent seasons in a year. Inducible promoters include promotersinduced by chemicals (e. g., exogenous or synthetic chemicals as well asendogenous pheromones and other signaling molecules) or by environmentalconditions such as, but not limited to, biotic or abiotic stress (e. g.,water deficit or drought, heat, cold, high or low nutrient or saltlevels, high or low light levels, or pest or pathogen infection). Anexpression-specific promoter can also include promoters that aregenerally constitutively expressed but at differing degrees or“strengths” of expression, including promoters commonly regarded as“strong promoters” or as “weak promoters”. The promoter element caninclude nucleic acid sequences that are not naturally occurringpromoters or promoter elements or homologues thereof but that canregulate expression of a gene. Examples of such “gene independent”regulatory sequences include naturally occurring or artificiallydesigned RNA sequences that include a ligand-binding region or aptamerand a regulatory region (which can be cis-acting). See, for example, thediscussion of RNA regulatory elements (“riboregulators”) given by Isaacset al. (2004) Nat. Biotechnol., 22:841-847, Bayer and Smolke (2005)Nature Biotechnol., 23:337-343, Mandal and Breaker (2004) Nature Rev.Mol. Cell Biol., 5:451-463, Davidson and Ellington (2005) TrendsBiotechnol., 23:109-112, Winkler et al. (2002) Nature, 419:952-956,Sudarsan et al. (2003) RNA, 9:644-647, and Mandal and Breaker (2004)Nature Struct. Mol. Biol., 11:29-35. Such “riboregulators” could beselected or designed for specific spatial or temporal specificity, forexample, to regulate translation of the exogenous gene only in thepresence (or absence) of a given concentration of the appropriateligand.

In some embodiments, a recombinant DNA construct containing thesuppression element includes both a promoter element and a functionalterminator element. Where it is functional, the terminator elementincludes a functional polyadenylation signal and polyadenylation site,allowing RNA transcribed from the recombinant DNA construct to bepolyadenylated and processed for transport into the cytoplasm. In otherembodiments, a functional terminator element is absent. In someembodiments where a functional terminator element is absent, at leastone of a functional polyadenylation signal and a functionalpolyadenylation site is absent. In other embodiments, a 3′ untranslatedregion is absent. In these cases, the recombinant DNA construct istranscribed as unpolyadenylated RNA and is preferably not transportedinto the cytoplasm.

In some embodiments, the suppression element is embedded within anintron, which is preferably an intron flanked on one or on both sides bynon-protein-coding DNA. One non-limiting embodiment is a recombinant DNAconstruct that consists entirely of non-coding DNA and that includes thesuppression element (or elements) embedded within an intron. Intronssuitable for use in constructs of the invention can be viral introns (e.g., Yamada et al. (1994) Nucleic Acids Res., 22:2532-2537), eukaryoticintrons (including animal, fungal, and plant introns), archeal orbacterial introns (e. g., Belfort et al. (1995) J. Bacteriol.,177:3897-3903), or any naturally occurring or artificial (e. g.,Yoshimatsu and Nagawa (1989) Science, 244:1346-1348) DNA sequences withintron-like functionality in the plant in which the recombinant DNAconstruct of the invention is to be transcribed. While essentially anyintron can be used in the practice of this invention as a host forembedded DNA, particularly preferred are introns that are introns thatenhance expression in a plant or introns that are derived from a 5′untranslated leader sequence. Where a recombinant DNA construct of theinvention is used to transform a plant, plant-sourced introns can beespecially preferred. Examples of especially preferred plant intronsinclude a rice actin 1 intron (I-Os-Act1) (Wang et al. (1992) Mol. CellBiol., 12:3399-3406; McElroy et al. (1990) Plant Cell, 2:163-171), amaize heat shock protein intron (I-Zm-hsp70) (U.S. Pat. Nos. 5,593,874and 5,859,347), and a maize alcohol dehydrogenase intron (I-Zm-adh1)(Callis et al. (1987) Genes Dev., 1:1183-1200). Other examples ofintrons suitable for use in the invention include the tobacco mosaicvirus 5′ leader sequence or “omega” leader (Gallie and Walbot (1992)Nucleic Acids Res., 20:4631-4638), the Shrunken-1 (Sh-1) intron (Vasilet al. (1989) Plant Physiol., 91:1575-1579), the maize sucrose synthaseintron (Clancy and Hannah (2002) Plant Physiol., 130:918-929), the heatshock protein 18 (hsp18) intron (Silva et al. (1987) J. Cell Biol.,105:245), and the 82 kilodalton heat shock protein (hsp82) intron(Semrau et al. (1989) J. Cell Biol., 109, p. 39A, and Mettler et al.(May 1990) N.A.T.O. Advanced Studies Institute on Molecular Biology,Elmer, Bavaria).

In some embodiments, the suppression element is included in arecombinant DNA construct that also includes at least one expressionelement (e. g., to express a gene of interest, a reporter gene, or amarker gene) or additional suppression elements.

Reference Suppression Elements and Modified Suppression Elements

The reference suppression element is any suppression element (see“Suppression Elements” beginning at paragraph 0025 above) used toestablish a reference degree of silencing (see “Degrees of Silencing”beginning at paragraph 0050 below). The modified suppression element isany suppression element that is different but derived from the referencesuppression element. Thus, modification of the reference suppressionelement to obtain one or more modified suppression elements can involvechanging the reference suppression element's size (e. g., byfragmentation or truncation), changing the reference suppressionelement's nucleotide sequence (e. g., by substitution or deletion ofnucleotides), changing the arrangement of the order and/or number ofreference suppression fragments, changing the reference suppressionelement's orientation (sense or anti-sense) or position in a recombinantDNA construct, or inclusion or deletion of stabilizing or destabilizingelements.

In one embodiment, the modified suppression element includes at leastone fragment of the reference suppression element. In some embodiments,the modified suppression element includes multiple fragments of thereference suppression element, which can be multiple different fragmentsof the reference suppression element or multiple copies of one or morefragments of the reference suppression element. Where the modifiedsuppression elements includes multiple different fragments of thereference suppression element, the multiple different fragments can bein the same order as, or in an order different from, that in which theyare arranged in the reference suppression element. In anotherembodiment, the modified suppression element is obtained by modificationof the suppression element to target one or more fragments (e. g.,truncations) of the target gene

At least one fragment of the reference suppression element may beprovided in a cell by any suitable means. In one embodiment, the atleast one fragment is obtained by truncation of the referencesuppression element. Such truncation includes deletion of one or morenucleotides from the 5′ end, from the 3′ end, or from both the 5′ and 3′ends of the reference suppression element. In another embodiment, the atleast one fragment is provided by deletion of one or more internal(non-terminal) nucleotides from the reference suppression element.Deletion can be of a single sequence of contiguous nucleotides from thereference suppression element, or of multiple such sequences.

Generally, the at least one fragment is at least 19 contiguousnucleotides in length, but the fragment can be any length necessary toobtain the desired degree of silencing, e. g., at least 21, at least 22,at least 23, or at least 24 nucleotides. In some embodiments, thefragment can include more than about 25, about 50, about 75, about 100,about 150, about 200, about 300, or about 500 nucleotides or greater. Incomparison to the length of the reference suppression element, the atleast one fragment can include between about 1% to about 98%, e. g.,about 98%, about 95%, about 90%, about 85%, about 80%, about 70%, about60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, orabout 1% of the length of the reference suppression element. The atleast one fragment can be less than 1% of the length of the referencesuppression element.

In one embodiment of the method, the modified suppression element isselected by mapping the silencing efficacy for the target gene. Mappingcan involve predictive methods or empirical determination of silencingefficacy. Predictive methods include, e. g., use of the criteria forpredicting the efficiency at which a given siRNA would function asidentified by Reynolds et al. (2004) Nature Biotechnol., 22:326-330, andfurther described in United States Patent Application Publication2005/0255487, which is incorporated by reference,. In one embodiment,mapping consists primarily or entirely of empirical determination ofsilencing efficacy. Empirical determination of silencing efficacy caninclude determining actual gene silencing (e. g., by measuring mRNA orprotein expression levels of the target gene or its encoded product), orany suitable proxy measurement. Proxy measurements can include, forexample, measuring a trait or phenotype that results from expression ofthe target gene (e. g., measuring the level of a metabolic product, ameasurable phenotype of a transgenic organism such as growth rate oryield or pest resistance of a transgenic plant).

Mapping can be carried out at any suitable resolution (in terms ofnucleotide length of a potential suppression element) of the targetgene. In general, fine mapping includes determination and/or predictionof efficacy of a minimum suppression element size, e. g., at least 19contiguous nucleotides in length, or at least 21, at least 22, at least23, or at least 24 nucleotides in length. However, mapping can becarried out for any length of suppression element, including suppressionelements that are larger than the reference suppression element.

In one embodiment, the modified selection element is further selected oradapted to avoid silencing of non-target genes, or to avoid generationof undesirable polypeptides, or both. Non-target genes can include anygene not intended to be silenced or suppressed, in the eukaryotic cellin which the suppression element is transcribed or in organisms that maycome into contact with the transcribed suppression element or itsproducts. In some embodiments (for example, where the suppressionelement is intended to have applications across multiple species), itcan be desirable for the suppression element to include sequence commonto multiple species in which the target gene is to be silenced. Thus,the suppression element can include nucleotide sequence selected to bespecific for one taxon (for example, specific to a genus, family, oreven a larger taxon such as a phylum, e. g., arthropoda) but not forother taxa (for example, plants or vertebrates or mammals). In onenon-limiting example, a suppression element includes nucleotide sequencefor dsRNA-mediated gene silencing in corn rootworm that is selected tobe specific to all members of the genus Diabrotica. In a furtherexample, such a Diabrotica-targetted suppression element is selected soas to not contain nucleotide sequence from beneficial coleopterans (forexample, predatory coccinellid beetles, commonly known as ladybugs orladybirds) or other beneficial insect species.

In another embodiment, the modified suppression element is furtherselected or adapted to avoid generation of undesirable polypeptides. Forexample, a suppression element can be screened to eliminate or minimizesequences that encode known undesirable polypeptides or close homologuesof these. Undesirable polypeptides include, but are not limited to,polypeptides homologous to known allergenic polypeptides andpolypeptides homologous to known polypeptide toxins. Sequences encodingsuch undesirable potentially allergenic peptides are known in the art(e. g., Gendel (1998) Adv. Food Nutr. Res., 42:63-92) and are publiclyavailable, for example, at the Food Allergy Research and ResourceProgram (FARRP) allergen database or the Biotechnology Information forFood Safety Databases. Undesirable sequences can also include, forexample, those polypeptide sequences annotated as known toxins or aspotential or known allergens and contained in publicly availabledatabases such as GenBank, EMBL, SwissProt, and others, which aresearchable by the Entrez system. Non-limiting examples of undesirable,potentially allergenic peptide sequences include glycinin from soybean,oleosin and agglutinin from peanut, glutenins from wheat, casein,lactalbumin, and lactoglobulin from bovine milk, and tropomysosin fromvarious shellfish. Non-limiting examples of undesirable, potentiallytoxic peptides include tetanus toxin tetA from Clostridium tetani,diarrheal toxins from Staphylococcus aureus, and venoms such asconotoxins from Conus spp. and neurotoxins from arthropods and reptiles.

In one non-limiting example, potential suppression element sequenceswere screened to eliminate those sequences encoding polypeptides withperfect homology to a known allergen or toxin over 8 contiguous aminoacids, or with at least 35% identity over at least 80 amino acids; suchscreens can be performed on any and all possible reading frames in bothdirections, on potential open reading frames that begin with ATG, or onall possible reading frames, regardless of whether they start with anATG or not.

In a non-limiting example, screens (referred to as “EAT/Tox” screens)were routinely performed on the transcribed portions of suppressionelements that are intended to produce a double stranded RNA but are notintended to be translated into a polypeptide. These screens can beperformed on any and all possible reading frames in both directions, andon potential open reading frames that begin with ATG, or on all possiblereading frames, regardless of whether they start with an ATG or not.When a “hit” or match is made, that is, when a sequence that encodes apotential polypeptide with perfect homology to a known allergen or toxinover 8 contiguous amino acids (or at least about 35% identity over atleast about 80 amino acids), is identified, the nucleotide sequencescorresponding to the hit can be avoided, eliminated, or modified whenselecting a suppression element.

Avoiding, elimination of, or modification of, an undesired sequence maybe achieved by any of a number of methods known to those skilled in theart. In some cases, the result may be novel sequences that are believedto not exist naturally. For example, avoiding certain sequences can beaccomplished by joining together “clean” sequences into novel chimericsuppression element that will produce a novel transcript, mostpreferably a transcript that provides the desired modified level ofsuppression.

Applicants recognize that it is possible for suppression elementsequences that imperfectly correspond to the intended target gene to beeffective at gene silencing. For example, it has been shown thatmismatches near the center of a miRNA complementary site has strongereffects on the miRNA's gene silencing than do more distally locatedmismatches (see, for example, FIG. 4 in Mallory et al. (2004) EMBO J.,23:3356-3364). In another example, it has been reported that, both theposition of a mismatched base pair and the identity of the nucleotidesforming the mismatch influence the ability of a given siRNA to silence atarget gene, and that adenine-cytosine mismatches, in addition to theG:U wobble base pair, were well tolerated, as described by Du et al.(2005) Nucleic Acids Res., 33:1671-1677. Thus, a suppression elementneed not always have 100% sequence identity with (or 100% complementaryto) the intended target gene, but generally would preferably havesubstantial sequence identity (or complementarity) with the intendedtarget gene, such as about 95%, about 90%, about 85%, or about 80%sequence identity with (or complementarity to) the intended target gene.One skilled in the art would be capable of judging the importance givento screening for regions predicted to be more highly specific to thetarget gene or predicted to not generate undesirable polypeptides,relative to the importance given to other criteria, such as, but notlimited to, the percent sequence identity with the intended target geneor the predicted or empirically determined silencing efficacy of a givensuppression element. For example, it may be desirable for a givensuppression element to be active across several species, and thereforeone skilled in the art may determine that it is more important toinclude regions specific to the several species of interest, but lessimportant to screen for regions predicted to have higher gene silencingefficiency or for regions predicted to generate undesirablepolypeptides.

Degrees of Silencing

Transcription in the eukaryotic cell of a reference suppression element(see “Reference Suppression Elements and Modified Suppression Elements”beginning at paragraph 0038 above) that targets a given target genepreferably results in the suppression of expression of the target geneat a certain level (termed the “reference degree of silencing”),relative to the level of expression of the target gene in the absence ofsuch transcription. The reference degree of silencing is not an absolutelevel of suppression but rather is a level of suppression selected as abenchmark for comparison with the level of suppression by alternativesuppression elements. Thus, the reference degree of silencing of thetarget gene can be any level of suppression, from about zero suppression(relative to expression in the absence of transcription of the referencesuppression element) to about complete suppression, or any level inbetween, and the degree of silencing can be selected as described hereinto achieve a desired level of modulation of expression of the targettedsequence (or target gene). Use of the term “reference degree ofsilencing” in this context is not meant to imply a “maximum” or“optimal” degree of silencing.

Transcription in the eukaryotic cell of a modified suppression element(see “Reference Suppression Elements and Modified Suppression Elements”beginning at paragraph 0038 above) preferably results in a level ofsuppression of expression of the target gene that differs from thatobtained by transcription of the reference suppression element, whereinsuch level of suppression is termed the “modified degree of silencing”.In some embodiments, the modified degree of silencing is increasedsilencing of the target gene, relative to the reference degree ofsilencing. In other embodiments, the modified degree of silencing isdecreased silencing of the target gene, relative to the reference degreeof silencing.

Detecting or measuring the degree of silencing of a target generesulting from transcription of the suppression element can be achievedby any suitable method to detect the expression (or suppression) of DNAsequence or the RNA transcript corresponding to the target gene, or of apeptide encoded by the target gene. Suitable methods include proteindetection methods (e. g., western blots, ELISAs, and otherimmunochemical methods, and measurements of enzymatic activity), ornucleic acid detection methods (e. g., Southern blots, northern blots,PCR, RT-PCR, fluorescent in situ hybridization, and TaqMAN assays). Suchmethods are well known to those of ordinary skill in the art asevidenced by the numerous handbooks available; see, for example, JosephSambrook and David W. Russell, “Molecular Cloning: A Laboratory Manual”(third edition), Cold Spring Harbor Laboratory Press, NY, 2001;Frederick M. Ausubel et al. (editors) “Short Protocols in MolecularBiology” (fifth edition), John Wiley and Sons, 2002; John M. Walker(editor) “Protein Protocols Handbook” (second edition), Humana Press,2002; and Leandro Peña (editor) “Transgenic Plants: Methods andProtocols”, Humana Press, 2004.

Other suitable methods for detecting or measuring suppression of atarget gene include measurement of any other trait that is a proxy(surrogate) indication of gene suppression in the eukaryotic cell inwhich the suppression element is transcribed, relative to a cell inwhich the modified or reference suppression element is not transcribed.Such proxy indications include, e. g., gross or microscopicmorphological traits, growth rates, yield, reproductive or recruitmentrates, resistance to pests or pathogens, or resistance to biotic orabiotic stress (e. g., water deficit stress, salt stress, nutrientstress, heat or cold stress). Proxy measurements of gene suppressioninclude measurements of a phenotypic trait (e. g., growth rates, levelsof a metabolite in a tissue, mortality in animals in which an animaltarget gene is suppressed) and in vitro assays (e. g., plant part assayssuch as leaf or root assays to indicate tolerance of abiotic stress).

Method of Providing a Desired Trait in a Eukaryote

In another aspect, the invention provides a method of providing aeukaryotic cell having a desired phenotype resulting from transcriptionin the eukaryotic cell of a modified suppression element, including (a)providing a range of modified suppression elements, wherein eachmodified suppression element includes a fragment of a referencesuppression element; (b) separately introducing each of the range ofmodified suppression elements into a eukaryotic cell, thereby producinga plurality of transgenic eukaryotic cells; (c) transcribing in each ofthe transgenic eukaryotic cell the modified suppression element thereinintroduced, and observing the resulting phenotype resulting from thetranscribing; and (d) selecting from the plurality of transgeniceukaryotic cells at least one eukaryotic cell having the desiredphenotype.

The range of modified suppression elements includes any suitable numberof modified suppression elements of one or more suppression elementtypes as described under “Suppression Elements” (beginning at paragraph0025 above) and “Reference Suppression Elements and Modified SuppressionElements” (beginning at paragraph 0038 above).

In one embodiment, the eukaryotic cell includes a transgenic plant cell.In some embodiments, the method further includes growing a transgenicplant including the transgenic plant cell. Suitable techniques forproducing a transgenic plant including the transgenic plant cell aredescribed under “Making and Using Transgenic Plant Cells and TransgenicPlants” beginning at paragraph 0057 below.

Making and Using Transgenic Plant Cells and Transgenic Plants

In preferred embodiments of the invention, the suppression istranscribed in a transgenic plant cell. The transgenic plant cell can bean isolated plant cell (e. g., individual plant cells or cells grown inor on an artificial culture medium), or can be a plant cell inundifferentiated tissue (e. g., callus or any aggregation of plantcells). The transgenic plant cell can be a plant cell in at least onedifferentiated tissue selected from the group consisting of leaf (e. g.,petiole and blade), root, stem (e. g., tuber, rhizome, stolon, bulb, andcorm) stalk (e. g., xylem, phloem), wood, seed, fruit (e. g., nut,grain, fleshy fruits), and flower (e. g., stamen, filament, anther,pollen, carpel, pistil, ovary, ovules). The invention further provides atransgenic plant having in its genome any of the reference or modifiedsuppression elements (or recombinant DNA constructs including thereference or modified suppression element) presently disclosed,including a regenerated plant prepared from the transgenic plant cellsdisclosed and claimed herein, or a progeny plant (which can be a hybridprogeny plant) of the regenerated plant, or seed of such a transgenicplant. Also provided is a transgenic seed having in its genome any ofthe reference or modified suppression elements or recombinant DNAconstructs presently disclosed, and a transgenic plant grown from suchtransgenic seed.

The transgenic plant cell or plant of the invention can be any plantcell or plant. Stably transformed transgenic plants are particularlypreferred. In many preferred embodiments, the transgenic plant is afertile transgenic plant from which seed can be harvested, and thus theinvention further claims seed of such transgenic plants, wherein theseed is preferably also transgenic, that is, preferably contains thereference or modified suppression elements or recombinant DNA constructsof the invention.

Where a recombinant DNA construct is used to produce a transgenic plantcell or transgenic plant of the invention, the transformation caninclude any of the well-known and demonstrated methods and compositions.Suitable methods for plant transformation include virtually any methodby which DNA can be introduced into a cell, such as by direct deliveryof DNA (e. g., by PEG-mediated transformation of protoplasts, byelectroporation, by agitation with silicon carbide fibers, and byacceleration of DNA coated particles), by Agrobacterium-mediatedtransformation, by viral or other vectors, etc. One preferred method ofplant transformation is microprojectile bombardment, for example, asillustrated in U.S. Pat. Nos. 5,015,580 (soy), 5,550,318 (maize),5,538,880 (maize), 6,153,812 (wheat), 6,160,208 (maize), 6,288,312(rice), 6,399,861 (maize), and 6,403,865 (maize).

Another preferred method of plant transformation isAgrobacterium-mediated transformation. In one preferred embodiment ofthe invention, the transgenic plant cell of the invention is obtained bytransformation by means of Agrobacterium containing a binary Ti plasmidsystem, wherein the Agrobacterium carries a first Ti plasmid and asecond, chimeric plasmid containing at least one T-DNA border of awild-type Ti plasmid, a promoter functional in the transformed plantcell and operably linked to a gene suppression construct of theinvention. See, for example, U.S. Pat. No. 5,159,135; De Framond (1983)Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. Insuch a binary system, the smaller plasmid, containing the T-DNA borderor borders, can be conveniently constructed and manipulated in asuitable alternative host, such as E. coli, and then transferred intoAgrobacterium.

Detailed procedures for Agrobacterium-mediated transformation of plants,especially crop plants, include, for example, procedures disclosed inU.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton); 5,416,011,5,569,834, 5,824,877 and 6,384,301 (soy); 5,591,616 (maize); 5,981,840(maize); and 5,463,174 (brassicas). Similar methods have been reportedfor, among others, peanut (Cheng et al. (1996) Plant Cell Rep., 15:653); asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. U.S.A.,84:5345); barley (Wan and Lemaux (1994) Plant Physiol., 104:37); rice(Toriyama et al. (1988) Bio/Technology, 6:10; Zhang et al. (1988) PlantCell Rep., 7:379; wheat (Vasil et al. (1992) Bio/Technology, 10:667;Becker et al. (1994) Plant J., 5:299), and alfalfa (Masoud et al. (1996)Transgen. Res., 5:313). See also United States Patent ApplicationPublication 2003/0167537A1 for a description of vectors, transformationmethods, and production of transformed Arabidopsis thaliana plants wheretranscription factors are constitutively expressed by a CaMV35Spromoter. Transgenic plant cells and transgenic plants can also beobtained by transformation with other vectors, such as, but not limitedto, viral vectors (e. g., tobacco etch potyvirus (TEV), barley stripemosaic virus (BSMV), and the viruses referenced in Edwardson andChristie, “The Potyvirus Group: Monograph No. 16, 1991, Agric. Exp.Station, Univ. of Florida), plasmids, cosmids, YACs (yeast artificialchromosomes), BACs (bacterial artificial chromosomes) or any othersuitable cloning vector used with an appropriate transformationprotocol, e. g., bacterial infection (e. g., with Agrobacterium asdescribed above), binary bacterial artificial chromosome constructs,direct delivery of DNA (e. g., via PEG-mediated transformation,desiccation/inhibition-mediated DNA uptake, electroporation, agitationwith silicon carbide fibers, and microprojectile bombardment). It wouldbe clear to one of skill in the art that various transformationmethodologies can be used and modified for production of stabletransgenic plants from any number of plant species.

Transformation methods to provide transgenic plant cells and transgenicplants containing stably integrated recombinant DNA are preferablypracticed in tissue culture on media and in a controlled environment.“Media” refers to the numerous nutrient mixtures that are used to growcells in vitro, that is, outside of the intact living organism.Recipient cell targets include, but are not limited to, meristem cells,callus, immature embryos or parts of embryos, and gametic cells such asmicrospores, pollen, sperm, and egg cells. It is contemplated that anycell from which a fertile plant can be regenerated can be useful as arecipient cell for practice of the invention. Callus can be initiatedfrom various tissue sources, including, but not limited to, immatureembryos or parts of embryos, seedling apical meristems, microspores, andthe like. Those cells which are capable of proliferating as callus canserve as recipient cells for genetic transformation. Practicaltransformation methods and materials for making transgenic plants ofthis invention (e. g., various media and recipient target cells,transformation of immature embryos, and subsequent regeneration offertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos.6,194,636 and 6,232,526 and United States Application Publication2004/0216189.

In general transformation practice, DNA is introduced into only a smallpercentage of target cells in any one transformation experiment. Markergenes are generally used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theantibiotics or herbicides to which a plant cell may be resistant can bea useful agent for selection. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells can be tested further to confirm stable integration of therecombinant DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin or paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(EPSPS). Examples of useful selective marker genes and selection agentsare illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and6,118,047. Screenable markers or reporters, such as markers that providean ability to visually identify transformants can also be employed.Non-limiting examples of useful screenable markers include, for example,a gene expressing a protein that produces a detectable color by actingon a chromogenic substrate (e. g., beta-glucuronidase (GUS) (uidA) orluciferase (luc)) or that itself is detectable, such as greenfluorescent protein (GFP) (gfp) or an immunogenic molecule. Those ofskill in the art will recognize that many other useful markers orreporters are available for use.

The recombinant DNA constructs of the invention can be stacked withother recombinant DNA for imparting additional traits (e. g., in thecase of transformed plants, traits including herbicide resistance, pestresistance, cold germination tolerance, water deficit tolerance, and thelike) for example, by expressing or suppressing other genes. Constructsfor coordinated decrease and increase of gene expression are disclosedin United States Patent Application Publication 2004/0126845 A1.

Seeds of transgenic, fertile plants can be harvested and used to growprogeny generations, including hybrid generations, of transgenic plantsthat include the reference or modified suppression elements orrecombinant DNA constructs in their genome. Thus, in addition to directtransformation of a plant with a recombinant DNA construct, transgenicplants can be prepared by crossing a first plant having the reference ormodified suppression elements or recombinant DNA constructs with asecond plant lacking the reference or modified suppression elements orrecombinant DNA constructs. For example, recombinant DNA can beintroduced into a plant line that is amenable to transformation toproduce a transgenic plant, which can be crossed with a second plantline to introgress the recombinant DNA into the resulting progeny. Atransgenic plant with the reference or modified suppression elements orrecombinant DNA constructs can be crossed with a plant line having otherrecombinant DNA that confers one or more additional trait(s) (such as,but not limited to, herbicide resistance, pest or disease resistance,environmental stress resistance, modified nutrient content, and yieldimprovement) to produce progeny plants having recombinant DNA thatconfers both the desired target sequence expression behavior and theadditional trait(s).

Typically, in such breeding for combining traits the transgenic plantdonating the additional trait is a male line and the transgenic plantcarrying the base traits is the female line. The progeny of this crosssegregate such that some of the plant will carry the DNA for bothparental traits and some will carry DNA for one parental trait; suchplants can be identified by markers associated with parental recombinantDNA Progeny plants carrying DNA for both parental traits can be crossedback into the female parent line multiple times, e. g., usually 6 to 8generations, to produce a progeny plant with substantially the samegenotype as one original transgenic parental line but for therecombinant DNA of the other transgenic parental line.

Yet another aspect of the invention is a transgenic plant grown from thetransgenic seed of the invention. This invention contemplates transgenicplants grown directly from transgenic seed containing the reference ormodified suppression elements or recombinant DNA constructs as well asprogeny generations of plants, including inbred or hybrid plant lines,made by crossing a transgenic plant grown directly from transgenic seedto a second plant not grown from the same transgenic seed.

Crossing can include, for example, the following steps:

(a) plant seeds of the first parent plant (e. g., non-transgenic or atransgenic) and a second parent plant that is transgenic according tothe invention;

(b) grow the seeds of the first and second parent plants into plantsthat bear flowers;

(c) pollinate a flower from the first parent with pollen from the secondparent; and

(d) harvest seeds produced on the parent plant bearing the fertilizedflower.

It is often desirable to introgress recombinant DNA into elitevarieties, e. g., by backcrossing, to transfer a specific desirabletrait from one source to an inbred or other plant that lacks that trait.This can be accomplished, for example, by first crossing a superiorinbred (“A”) (recurrent parent) to a donor inbred (“B”) (non-recurrentparent), which carries the appropriate gene(s) for the trait inquestion, for example, a construct prepared in accordance with thecurrent invention. The progeny of this cross first are selected in theresultant progeny for the desired trait to be transferred from thenon-recurrent parent “B”, and then the selected progeny are mated backto the superior recurrent parent “A”. After five or more backcrossgenerations with selection for the desired trait, the progeny arehemizygous for loci controlling the characteristic being transferred,but are like the superior parent for most or almost all other genes. Thelast backcross generation would be selfed to give progeny which are purebreeding for the gene(s) being transferred, i. e., one or moretransformation events.

Through a series of breeding manipulations, a selected DNA construct canbe moved from one line into an entirely different line without the needfor further recombinant manipulation. One can thus produce inbred plantswhich are true breeding for one or more DNA constructs. By crossingdifferent inbred plants, one can produce a large number of differenthybrids with different combinations of DNA constructs. In this way,plants can be produced which have the desirable agronomic propertiesfrequently associated with hybrids (“hybrid vigor”), as well as thedesirable characteristics imparted by one or more DNA constructs.

Genetic markers can be used to assist in the introgression of one ormore DNA constructs of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers can provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers can be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized. The usefulness ofmarker assisted selection in breeding transgenic plants of the currentinvention, as well as types of useful molecular markers, such as but notlimited to SSRs and SNPs, are discussed in PCT Application PublicationWO 02/062129 and United States Patent Application Publications Numbers2002/0133852, 2003/0049612, and 2003/0005491.

In certain transgenic plant cells and transgenic plants of theinvention, it may be desirable to concurrently express (or suppress) agene of interest while also regulating expression of a target gene.Thus, in some embodiments, the transgenic plant contains recombinant DNAfurther including a gene expression (or suppression) element forexpressing at least one gene of interest, and regulation of expressionof a target gene is preferably effected with concurrent expression (orsuppression) of the at least one gene of interest in the transgenicplant.

Thus, as described herein, the transgenic plant cells or transgenicplants can be obtained by use of any appropriate transient or stable,integrative or non-integrative transformation method known in the art orpresently disclosed. The reference or modified suppression elements orrecombinant DNA constructs can be transcribed in any plant cell ortissue or in a whole plant of any developmental stage. Transgenic plantscan be derived from any monocot or dicot plant, such as, but not limitedto, plants of commercial or agricultural interest, such as crop plants(especially crop plants used for human food or animal feed), wood- orpulp-producing trees, vegetable plants, fruit plants, and ornamentalplants. Non-limiting examples of plants include grain crop plants (suchas wheat, oat, barley, maize, rye, triticale, rice, millet, sorghum,quinoa, amaranth, and buckwheat); forage crop plants (such as foragegrasses and forage dicots including alfalfa, vetch, clover, and thelike); oilseed crop plants (such as cotton, safflower, sunflower,soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (suchas walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane,coconut, date palm, olive, sugarbeet, tea, and coffee; wood- orpulp-producing trees; vegetable crop plants such as legumes (forexample, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus,artichoke, celery, carrot, radish, the brassicas (for example, cabbages,kales, mustards, and other leafy brassicas, broccoli, cauliflower,Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example,cucumbers, melons, summer squashes, winter squashes), edible alliums(for example, onions, garlic, leeks, shallots, chives), edible membersof the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers,groundcherries), and edible members of the Chenopodiaceae (for example,beet, chard, spinach, quinoa, amaranth); fruit crop plants such asapple, pear, citrus fruits (for example, orange, lime, lemon,grapefruit, and others), stone fruits (for example, apricot, peach,plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado,and berries; and ornamental plants including ornamental floweringplants, ornamental trees and shrubs, ornamental groundcovers, andornamental grasses. Preferred dicot plants include, but are not limitedto, canola, cotton, potato, quinoa, amaranth, buckwheat, safflower,soybean, sugarbeet, and sunflower, more preferably soybean, canola, andcotton. Preferred monocots include, but are not limited to, wheat, oat,barley, maize, rye, triticale, rice, ornamental and forage grasses,sorghum, millet, and sugarcane, more preferably maize, wheat, and rice.

The ultimate goal in plant transformation is to produce plants which areuseful to man. In this respect, transgenic plants of the invention canbe used for virtually any purpose deemed of value to the grower or tothe consumer. For example, one may wish to harvest the transgenic plantitself, or harvest transgenic seed of the transgenic plant for plantingpurposes, or products can be made from the transgenic plant or its seedsuch as oil, starch, ethanol or other fermentation products, animal feedor human food, pharmaceuticals, and various industrial and commodityproducts. For example, maize is used extensively in the food and feedindustries, as well as in industrial applications. Further discussion ofthe uses of maize can be found, for example, in U.S. Pat. Nos.6,194,636, 6,207,879, 6,232,526, 6,426,446, 6,429,357, 6,433,252,6,437,217, and 6,583,338 and PCT Publications WO 95/06128 and WO02/057471.

Thus, in preferred embodiments, the suppression element is transcribedin a transgenic plant cell contained in a transgenic plant or planttissue, resulting in at least one altered trait, relative to a plant orplant tissue lacking the suppression element, selected from the group oftraits consisting of:

(a) improved abiotic stress tolerance;

(b) improved biotic stress tolerance;

(c) improved resistance to a pest or pathogen of the plant;

(d) modified primary metabolite composition;

(e) modified secondary metabolite composition;

(f) modified trace element, carotenoid, or vitamin composition;

(g) improved yield;

(h) improved ability to use nitrogen or other nutrients;

(i) modified agronomic characteristics;

(j) modified growth or reproductive characteristics; and

(k) improved harvest, storage, or processing quality.

In particularly preferred embodiments, the transgenic plant cell, planttissue, or plant is characterized by: improved tolerance of abioticstress (e. g., tolerance of water deficit or drought, heat, cold,non-optimal nutrient or salt levels, non-optimal light levels) or ofbiotic stress (e. g., crowding, allelopathy, or wounding); by improvedresistance to a pest or pathogen (e. g., insect, nematode, fungal,bacterial, or viral pest or pathogen) of the plant; by a modifiedprimary metabolite (e. g., fatty acid, oil, amino acid, protein, sugar,or carbohydrate) composition; a modified secondary metabolite (e. g.,alkaloids, terpenoids, polyketides, non-ribosomal peptides, andsecondary metabolites of mixed biosynthetic origin) composition; amodified trace element (e. g., iron, zinc), carotenoid (e. g.,beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids andxanthophylls), or vitamin (e. g., tocopherols) composition; improvedyield (e. g., improved yield under non-stress conditions or improvedyield under biotic or abiotic stress); improved ability to use nitrogenor other nutrients; modified agronomic characteristics (e. g., delayedripening; delayed senescence; earlier or later maturity; improved shadetolerance; improved resistance to root or stalk lodging; improvedresistance to “green snap” of stems; modified photoperiod response);modified growth or reproductive characteristics (e. g., intentionaldwarfing; intentional male sterility, useful, e. g., in improvedhybridization procedures; improved vegetative growth rate; improvedgermination; improved male or female fertility); improved harvest,storage, or processing quality (e. g., improved resistance to pestsduring storage, improved resistance to breakage, improved appeal toconsumers); or any combination of these traits.

In one preferred embodiment, transgenic seed, or seed produced by thetransgenic plant, has modified primary metabolite (e. g., fatty acid,oil, amino acid, protein, sugar, or carbohydrate) composition, amodified secondary metabolite (e. g., alkaloids, terpenoids,polyketides, non-ribosomal peptides, and secondary metabolites of mixedbiosynthetic origin) composition, a modified trace element (e. g., iron,zinc), carotenoid (e. g., beta-carotene, lycopene, lutein, zeaxanthin,or other carotenoids and xanthophylls), or vitamin (e. g., tocopherols,)composition, an improved harvest, storage, or processing quality, or acombination of these. For example, it can be desirable to modify theamino acid (e. g., lysine, methionine, tryptophan, or total protein),oil (e. g., fatty acid composition or total oil), carbohydrate (e. g.,simple sugars or starches), trace element, carotenoid, or vitamincontent of seeds of crop plants (e. g., canola, cotton, safflower,soybean, sugarbeet, sunflower, wheat, maize, or rice), preferably incombination with improved seed harvest, storage, or processing quality,and thus provide improved seed for use in animal feeds or human foods.In another instance, it can be desirable to change levels of nativecomponents of the transgenic plant or seed of a transgenic plant, forexample, to decrease levels of proteins with low levels of lysine,methionine, or tryptophan, or to increase the levels of a desired aminoacid or fatty acid, or to decrease levels of an allergenic protein orglycoprotein (e. g., peanut allergens including ara h 1, wheat allergensincluding gliadins and glutenins, soy allergens including P34 allergen,globulins, glycinins, and conglycinins) or of a toxic metabolite (e. g.,cyanogenic glycosides in cassava, solanum alkaloids in members of theSolanaceae).

EXAMPLES Example 1

This example illustrates a non-limiting example of a method of modifyingthe degree of silencing of a target gene, including transcribing in aeukaryotic cell a, thereby obtaining a modified degree of silencing ofthe target gene, relative to a modified suppression element referencedegree of silencing obtained through the transcription in the eukaryoticcell of a reference suppression element that corresponds to the targetgene. More particularly, this example describes a method includingselection of a modified suppression element by mapping silencingefficacy.

Mapping the silencing efficacy of suppression elements for the targetgene can be carried out, for example, by empirically determining thesilencing efficacy of each of a plurality of fragments of a givenreference suppression element that corresponds to the target gene. Oneembodiment of the method includes “scanning” the reference suppressionelement, that is, determining the silencing efficacy of fragments of thereference suppression element. The first fragments can be contiguous ornon-contiguous, and can overlap. Generally, each of the first fragmentsincludes from about 1% to about 98% of the reference suppressionelement; however, the reference suppression element may be divided intoas many first fragments as is convenient or desirable. One non-limitingexample of scanning includes the steps of: (a) providing a referencesuppression element corresponding to the target gene, whereintranscription of the reference suppression element in a eukaryotic cellresults in a reference degree of silencing of the target gene; (b)providing a plurality of first fragments of the reference suppressionelement; and (c) empirically determining the degree of silencing of thetarget gene for each of the first fragments when transcribed in theeukaryotic cell; and (d) selecting at least one first fragment thatprovides a desired degree of silencing of the target gene for use in amodified suppression element.

Another non-limiting example of scanning includes the steps of: (a)providing a reference suppression element corresponding to the targetgene, wherein transcription of the reference suppression element in aeukaryotic cell results in a reference degree of silencing of the targetgene; (b) providing a plurality of first fragments of the referencesuppression element; (c) empirically determining the degree of silencingof the target gene for each of the first fragments when transcribed inthe eukaryotic cell; (d) selecting at least one first fragment thatprovides a desired degree of silencing of the target gene for use in amodified suppression element; (e) for the selected at least one firstfragment, providing a plurality of second fragments each consisting ofnucleotide segments of the first fragment; empirically determining thedegree of silencing of the target gene for each of the second fragmentswhen transcribed in the eukaryotic cell; and (f) selecting at least onesecond fragment that provides a desired degree of silencing of thetarget gene for use in a modified suppression element. The fragments canbe contiguous or non-contiguous, and can overlap. The second fragmentsconsist of from about one nucleotide shorter than the at least one firstfragment to about 21 nucleotides in length. Fragments thus derived canbe combined, e. g., as multiple copies of one or more fragments inchimeric combinations, or in combination with other suppression elementsor expression elements.

The method of the invention is further illustrated by the followingexample, which describes mapping silencing efficacy of suppressionelements for the target gene corn rootworm V-ATPase subunit A byanalysis of fragments of a reference suppression element (FIG. 1). Abioassay for larval mortality served as a proxy measurement of targetgene silencing efficacy. The reference suppression element, a ˜600nucleotide segment of Western corn rootworm (“WCR”, Diabroticavirgifera) V-ATPase subunit A was found to induce a reference degree ofsilencing of ˜79% mortality when fed as double-stranded RNA (0.02 ppm)to WCR larvae. Four contiguous ˜150 nucleotide fragments of this ˜600nucleotide V-ATPase segment were separately synthesized and fed as dsRNA(0.02 ppm) to WCR larvae; these modified suppression elements were foundto induce modified degrees of suppression of 18%, 75%, 50%, and 12%mortality, respectively (FIG. 1). Thus, the modified degree ofsuppression resulting from transcription in a eukaryotic cell (e. g., ina transgenic corn plant cell developed for resistance to corn rootworm)of these ˜150 nucleotide modified suppression elements is decreasedsuppression (e. g., decreased WCR larval mortality), relative to thesuppression observed for the reference suppression element.

Another suppression element, consisting of a 100 nucleotide subfragmentof the ˜150 nucleotide fragment that induced 75% mortality at 0.02 ppm(FIG. 1), was selected for further efficacy mapping. The 100 base pairfragment was amplified by PCR with the appropriate primers to produce anantisense template and a sense template. The sense and antisensereactions were mixed, heated to 75 degrees Celsius for 5 minutes andallowed to cool to room temperature. The resulting annealed 100 basepair double-stranded RNA product was purified with the MEGAscript™ RNAiKit (Ambion, Cat #1626) according to the manufacturer's instructions toproduce a 100 base pair dsRNA product which was then tested with thesame WCR larval bioassay. When fed to WCR larvae at 0.2 ppm, the 100 bpdsRNA suppression element induced 100% mortality (FIG. 1). A control(double-stranded RNA derived from 108 base pairs of vector sequence)caused no mortality at the same feeding concentration.

The 100 bp suppression element was further mapped at a resolution of 26bp fragments in a 5 bp register (FIG. 1). Fifteen 26 bp segments(designated scans 0 to 14) derived from the 100 bp suppression elementwere produced synthetically (Integrated DNA Technologies) as sense andantisense oligonucleotides. Each pair of sense and antisenseoligonucleotides was annealed, and a 3′ overhang was added by PCR withREDtaq polymerase. The fifteen cloned 26 bp segments were verified forcorrect sequence and orientation, and used as PCR templates. PCRreaction products were checked on agarose gels for correct size andquality, and amplified for dsRNA synthesis using MEGAscript RNAi Kit(Ambion, Cat #1626). Final dsRNA products were quantified by absorptionat 260 nanometers, and visualized on a 1-3% agarose gel to ensureintactness of the product.

Double-stranded RNA corresponding to the fifteen 26 bp fragments (Scan 0to Scan 14, see Table 1 and FIG. 1) was amplified in a larger neutralcarrier (vector backbone sequence), and dsRNA was synthesized for atotal assayed dsRNA length of 206 bp. All samples for insect bioassaywere diluted to the final desired concentration in 10 millimolar Tris pH6.8. Twenty microliters of each sample were applied to 200 microlitersof insect diet and allowed to absorb into the diet before addition of aWCR neonate. Stunting and mortality of larvae was scored at day 12. Whenfed at 1 ppm, the dsRNAs synthesized from the 26 bp fragments resultedin a range of mortality from no significant difference from theuntreated control to approximately 95% mortality (Scan 7 fragment). Alower dose of 0.2 ppm was useful in identifying the most activesegments, with observed mortality ranging from no significant differencefrom the untreated control (Scan 10 fragment) to 97% mortality (Scan 3fragment) (Table 1 and FIG. 1).

TABLE 1 26mer mapping Larval mortality¹ in Larval mortality¹ in WCR dietbioassay at WCR diet bioassay at dsRNA 1 ppm 0.2 ppm Scan 0 60.1 ± 4.4 *13.3 ± 9.7 Scan 1  36.4 ± 16.3 * 16.3 ± 4.3 Scan 2 35.8 ± 9.1 * 22.6Scan 3 85.7 ± 9.0 * 96.7 ± 3.30 * Scan 4 75.0 ± 9.4 * 42.8 ± 3.8 * Scan5  65.4 ± 11.4 * 39.4 ± 10.7 * Scan 6 92.5 ± 5.0 * 61.9 ± 8.5 * Scan 794.6 ± 3.3 * 80.6 ± 9.4 * Scan 8  91.0 ± 5.61 * 66.7 ± 10.0 * Scan 941.4 ± 6.8 * 19.0 ± 7.5 Scan 10 7.9 ± 5.1  6.7 ± 4.1 Scan 11 39.3 ±5.3 * 5.4 ± 3.3 Scan 12 37.9 ± 6.9 * 13.7 ± 6.9 Scan 13 61.2 ± 6.3 *33.3 ± 12.6 * Scan 14 70.6 ± 7.3 * 42.3 ± 7.8 * 100 bp 100 * 100 *suppression element Control, vector NA  0.0 * sequence only ¹Percentmortality and standard error of the means. * significantly differentfrom untreated control, P value <0.05, Planned Contrasts. NA = notassayed

Scan 14 (26 bp) was further mapped into its seven possible 21 bpfragments (designated scans 15-21) (Table 2 and FIG. 1). Attempts weremade to clone all seven possible 21 mers, but cloning of scan 15 failedand the cloned scan 17 sequence was found to contain a point mutation.Nonetheless, the successfully cloned 21 mers were amplified to producetemplates, and embedded in neutral (vector) carrier sequence to give afinal dsRNA size of 184 bp. Samples were diluted, applied at 0.2 ppm,and assayed with the WCR larval diet bioassay as previously done. Mostwere found to possess significant activity (Table 2). Of particularinterest is the unpredicted discrepancy between the observed silencingefficacy (indicated by larval mortality) and the Reynolds scores of thetested fragments. A higher positive Reynolds score (Reynolds et al.2004) has been believed to be predictive a greater probability of genesuppression. Unexpectedly, the fragment with the highest Reynolds score(Scan 21, Table 2) gave the lowest activity, and the fragment with thelowest Reynolds score (Scan 16, Table 2), provided the highest activityof the tested 21 mers. These observed discrepancies emphasize the needfor empirical testing in mapping efficacy of suppression elements.

TABLE 2 21mer mapping Larval mortality¹ Larval mortality¹ in WCR in WCRReynolds score diet bioassay diet bioassay dsRNA (for 21mer) at 1 ppm at0.2 ppm Scan 14 (see not applicable 92.0 ± 8.0 * 77.3 ± 7.6 * Table 1)Scan 15 3 NA NA Scan 16 1 92.1 ± 5.1 * 53.2 ± 7.9 * Scan 17 3 13.6 ± 6.0  0.0 Scan 18 4  77.8 ± 10.0 * 43.2 ± 9.2 * Scan 19 6 73.3 ± 7.3 * 76.1± 9.6 * Scan 20 8 85.3 ± 6.2 * 77.1 ± 7.1 * Scan 21 9 5.0 ± 5.0  0.0 100bp not applicable 97.1 ± 2.9 * NA suppression element Control, vectornot applicable 0.0 NA sequence only ¹Percent mortality and standarderror of the means. * significantly different from untreated control, Pvalue <0.05, Planned Contrasts. NA = not assayed

Example 2

This example illustrates a non-limiting example of modifying the degreeof silencing of a target gene, including transcribing in a eukaryoticcell a modified suppression element, thereby obtaining a modified degreeof silencing of the target gene, relative to a reference degree ofsilencing obtained through the transcription in the eukaryotic cell of areference suppression element that corresponds to the target gene. Moreparticularly, this example describes a modified suppression elementprovided by truncation of a reference suppression element and a modifieddegree of silencing as evidenced by a modified phenotype in a seedcontaining the eukaryotic cell.

Furthermore, this example illustrates a method of providing a eukaryoticcell having a desired phenotype resulting from transcription in theeukaryotic cell of a modified suppression element. In one embodiment,the method includes the steps of: (a) providing a range of modifiedsuppression elements, wherein each modified suppression element includesa fragment of a reference suppression element; (b) separatelyintroducing each of the modified suppression elements into a eukaryoticcell, thereby producing a plurality of transgenic eukaryotic cells (c)transcribing in each of the transgenic eukaryotic cells the modifiedsuppression element therein introduced, and observing the resultingphenotype resulting from the transcribing; and (d) selecting from theplurality of transgenic eukaryotic cells at least one eukaryotic cellhaving the desired phenotype. In this non-limiting example, theeukaryotic cell is a plant cell, specifically a transgenic crop plantcell, the target gene includes non-coding sequence, the modifiedsuppression elements are a series of truncations of a referencesuppression element, and the desired phenotype is observed in a seedincluding the transgenic crop plant cell.

The target gene in this example is non-coding sequence, an intron of asoybean (Glycine max) fatty acid desaturase FAD2 or delta-12 desaturasegene, which encodes an enzyme that catalyzes the insertion of a doublebond into a monounsaturated 18:1 fatty acid to form a polyunsaturated18:2 fatty acid. Suppression of FAD2 results in an increase of 18:1fatty acid content in the seed.

To obtain a desired level of 18:1 fatty acids, the degree of silencingof the target gene was modified by use of modified suppression elements.Examples of recombinant DNA constructs, including constructs containingsuppression elements targetting FAD2 sequences, and detaileddescriptions of their use, as well as of methods to make and usetransgenic soybean seed, are provided in United States PatentApplication Publication 2004/0107460, which is incorporated by referencein its entirety herein. A reference suppression element includingsequence that transcribed to double-stranded RNA corresponding to theFAD2 intron was introduced into soybean by Agrobacterium-mediated stabletransformation, resulting in increased 18:1 fatty acid content. A rangeof modified suppression elements was designed, wherein each modifiedsuppression element was derived by truncation of the referencesuppression element to correspond respectively to a deletion of ˜160,˜240, or ˜320 nucleotides from the 5′ end of the target gene (FAD2intron). Each modified suppression element was introduced into soybeancells by Agrobacterium-mediated stable transformation, and stablytransformed soybean plants generated. FIG. 2 depicts the results oftranscribing the modified suppression element in soybean, where, onaverage, the degree of FAD2 silencing (observed as increased 18:1 fattyacid levels) was modulated or decreased in comparison to that obtainedwith the reference suppression element. Fatty acid content of individualseeds (six seeds per transformation event, with each event indicated onthe x-axis of FIG. 2) was analyzed by gas chromatography. Silencing ofthe target gene FAD2 (evidenced by increased 18:1 fatty acid content)was modified or decreased, with modulation (decrease in suppression)generally proportional to the degree of truncation (deletion of sequencefrom the reference suppression element) used in a given modifiedsuppression element, e. g., the increase in average 18:1 fatty acidcontent was less in seeds transformed with the 320-nucleotide truncatedmodified suppression element than in seeds transformed with the240-nucleotide truncated modified suppression element. Seed having thedesired phenotype (i. e., a given 18:1 fatty acid content) could then beselected from the range of phenotypes presented.

A separate experiment (FIG. 3) was carried out with modified suppressionelements wherein each modified suppression element was derived bytruncation of the reference suppression element to correspondrespectively to a deletion of ˜160, ˜240, or ˜320 nucleotides from the5′ end of the target gene (FAD2 intron) or to a deletion of ˜200, ˜240,˜260, ˜300, ˜320, ˜360, ˜380, ˜400 nucleotides from the 3′ end of thetarget gene (FAD2 intron). A suppression element corresponding tocomplete deletion (421 nucleotides) of the target gene (FAD2 intron)served as a control. Various, modified degrees of silencing of FAD2(evidenced by increased 18:1 fatty acid content) were obtained in thedifferent transgenic events, when compared to that obtained with theunmodified reference suppression element (“0 No”). Seed having thedesired phenotype (i. e., a given 18:1 fatty acid content) could then beselected from the range of phenotypes presented.

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. Although the materials and methods of this invention havebeen described in terms of preferred embodiments and illustrativeexamples, it will be apparent to those of skill in the art thatvariations can be applied to the materials and methods described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A method of modifying the degree of silencing ofa target gene, comprising transcribing in a eukaryotic cell a modifiedsuppression element, thereby obtaining a modified degree of silencing ofsaid target gene, relative to a reference degree of silencing obtainedthrough the transcription in said eukaryotic cell of a referencesuppression element that corresponds to said target gene.
 2. The methodof claim 1, wherein said target gene is endogenous to said eukaryoticcell.
 3. The method of claim 1, wherein said target gene is exogenous tosaid eukaryotic cell.
 4. The method of claim 1, wherein said target genecomprises one contiguous nucleotide sequence.
 5. The method of claim 1,wherein said target gene comprises non-contiguous nucleotide sequences.6. The method of claim 1, wherein said eukaryotic cell is selected froman animal cell and a plant cell.
 7. The method of claim 1, wherein saidmodified and reference suppression elements transcribe to at leastpartially double-stranded RNA.
 8. The method of claim 1, wherein saidmodified and reference suppression elements comprise at least one of:(a) DNA that comprises at least one anti-sense DNA segment that isanti-sense to at least one segment of said target gene; (b) DNA thatcomprises multiple copies of at least one anti-sense DNA segment that isanti-sense to at least one segment of said target gene; (c) DNA thatcomprises at least one sense DNA segment that is at least one segment ofsaid target gene; (d) DNA that comprises multiple copies of at least onesense DNA segment that is at least one segment of said target gene; (e)DNA that transcribes to RNA for suppressing said target gene by formingdouble-stranded RNA and comprises at least one anti-sense DNA segmentthat is anti-sense to at least one segment of said target gene and atleast one sense DNA segment that is at least one segment of said targetgene; (f) DNA that transcribes to RNA for suppressing said target geneby forming a single double-stranded RNA and comprises multiple serialanti-sense DNA segments that are anti-sense to at least one segment ofsaid target gene and multiple serial sense DNA segments that are atleast one segment of said target gene; (g) DNA that transcribes to RNAfor suppressing said target gene by forming multiple double strands ofRNA and comprises multiple anti-sense DNA segments that are anti-senseto at least one segment of said target gene and multiple sense DNAsegments that are at least one segment of said target gene, and whereinsaid multiple anti-sense DNA segments and said multiple sense DNAsegments are arranged in a series of inverted repeats; (h) DNA thatcomprises nucleotides derived from a plant miRNA; (i) DNA that comprisesnucleotides of a siRNA; (j) DNA that transcribes to an RNA aptamercapable of binding to a ligand; and (k) DNA that transcribes to an RNAaptamer capable of binding to a ligand, and DNA that transcribes toregulatory RNA capable of regulating expression of said target gene,wherein said regulation is dependent on the conformation of saidregulatory RNA, and said conformation of said regulatory RNA isallosterically affected by the binding state of said RNA aptamer
 9. Themethod of claim 1, wherein said modified degree of silencing isincreased silencing, relative to said reference degree of silencing 10.The method of claim 1, wherein said modified degree of silencing isdecreased silencing, relative to said reference degree of silencing 11.The method of claim 1, wherein said modified suppression elementcomprises at least one fragment of said reference suppression element.12. The method of claim 11, wherein said at least one fragment isobtained by truncation of said reference suppression element.
 13. Themethod of claim 11, wherein said at least one fragment comprises atleast about 1% of said reference suppression element.
 14. The method ofclaim 1, wherein said modified suppression element is selected bymapping the silencing efficacy for the target gene.
 15. The method ofclaim 1, wherein said modified suppression element is further selectedto avoid silencing of non-target genes.
 16. The method of claim 1,wherein said modified suppression element is further selected to avoidgeneration of undesirable polypeptides.
 17. A method of providing aeukaryotic cell having a desired phenotype resulting from transcriptionin said eukaryotic cell of a modified suppression element, comprising:(a) providing a range of modified suppression elements, wherein eachmodified suppression element comprises a fragment of a referencesuppression element; (b) separately introducing each of said modifiedsuppression elements into a eukaryotic cell, thereby producing aplurality of transgenic eukaryotic cells; (c) transcribing in each ofsaid transgenic eukaryotic cells the modified suppression elementtherein introduced, and observing the resulting phenotype resulting fromsaid transcribing; and (d) selecting from said plurality of transgeniceukaryotic cells at least one eukaryotic cell having said desiredphenotype.
 18. The method of claim 17, wherein said eukaryotic cellcomprises a transgenic plant cell.